Specific vertebrate cone opsins with distinct peak spectral sensitivities are expressed in separate cone populations, which provide differential input to downstream neurons. This information is interpreted as color. In humans, the opsins that sense red and green light (LWS/MWS) are encoded by an array of tandemly replicated genes sharing a single locus control region (L/M array). Defects in this array result in color blindness and more severe cone degenerative disorders. Zebrafish possess an orthologous tandemly replicated long wavelength- sensitive array (lws array, red-sensing). The process by which tandemly replicated opsins are differentially regulated is poorly understood, and filling this knowledge gap would allow the vision science community to develop treatments for disorders related to defects in the L/M array, as well as establish conditions for differentiating human cones in vitro for cell replacement therapies that permit high acuity color vision. The prevalent model for control of the human array predicts stochastic regulation. However, the presence of topographic gradients in LWS:MWS cone ratios in human retinas (and LWS1:LWS2 ratios in zebrafish) suggests that a trans regulatory mechanism is involved. The Stenkamp lab demonstrated that thyroid hormone (TH) regulates opsin expression in LWS cones, supporting the presence of a trans regulatory mechanism. The proposed research aims to characterize TH-mediated biological differences between the LWS cones and to investigate the genetic mechanisms underlying the switch in LWS cone opsin expression upon TH exposure in zebrafish. We will use single-cell RNA sequencing (scRNA-Seq) to obtain comprehensive transcriptional signatures of LWS cones from control and TH-treated zebrafish. We will also use previously collected bulk RNA-Seq and single-cell RNA-Seq (scRNA-Seq) data to identify genes that are differentially expressed (DE) in LWS1 vs LWS2 cones, and, after validation, we will determine whether the candidate genes are coordinately regulated by TH. Thirdly, we will use CRISPR/Cas9-mediated cell type-selective gene disruption strategies to elucidate the roles of TH receptors (TRs) and retinoid X receptors (RXRs) in TH-induced opsin switching. This project will provide insight into zebrafish LWS cone differentiation and ultimately into the mechanisms by which cones expressing one member of a tandemly duplicated opsin gene array differentiate in general.